大鼠骨髓基质细胞在体外条件诱导下向Schwann细胞分化的基础研究

发布时间：2018-07-21 14:40

【摘要】： 迄今为止,周围神经缺损的修复仍然是临床外科领域尚待解决的疑难问题之一。如果两断端距离过远则影响再生轴突的生长。临床上常用神经移植进行修补,虽然传统的自体神经移植方法效果最好,但因为材料来源的限制而制约了它的应用范围;而同种异体神经移植又由于免疫排斥、移植成功率低等原因而难以推广。20世纪以来,神经组织工程研究的发展,尤其是由支架材料和细胞外基质、种子细胞以及诱导和促进生长的因子等几个部分组成的“组织工程神经”的构建为人们寻找神经代用品修复周围神经缺损带来了新的希望。 Schwann细胞是神经组织工程中最重要、也是最常使用的种子细胞。但由于自体Schwann细胞来源有限且不容易增殖;而异体的Schwann细胞又会引起免疫排斥反应,因此将Schwann细胞化的“组织工程神经”应用于临床的研究仍遇到难以逾越的障碍。骨髓基质细胞(bone marrow stromal cells MSCs)因取材方便,来源广泛,在一定培养条件下可迅速大量扩增,具有自我更新、活跃增殖和多向分化潜能,且自体移植克服了伦理和免疫排斥等问题,作为一种理想的种子细胞脱颖而出。近年来一些在体外的研究证明MSCs在给予合适的诱导条件下,可以表达神经元的标记。这些研究表明MSCs在诱导分化后形态上发生了类似神经元的形态变化并且可以表达神经细胞的特异性蛋白,所以这些研究者认为是MSCs向神经细胞发生了转分化。但是也有持不同观点的报道,有研究者认为MSCs在体内的分化是与宿主的神经系细胞发生融合的过程。有的研究认为MSCs并没有真正的分化,而细胞形态的改变仅是由于诱导剂的化学性刺激的作用引起细胞的收缩和细胞骨架的塌陷。因此,目前关于MSCs是否有潜能分化成神经系的细胞是当今干细胞研究领域中的一个热点问题。我们以前的研究证明了大鼠的MSCs移植到坐骨神经受损的部位后,在体内有一部分能够分化为Schwann细胞样细胞并且表达S100蛋白,MSC移植到体内是有利于神经的再生。为了进一步分析MSC在向Schwann细胞分化的过程中Schwann细胞标记蛋白S100蛋白是否真正发生了表达变化,我们的实验探讨大鼠骨髓基质细胞在体外诱导的过程中,MSCs的形态变化以及表达Schwann细胞的标记蛋白S100B蛋白的变化。我们从成年SD大鼠的股骨和胫骨中分离培养MSCs。采用MTT法检测细胞的活力,FCM检测细胞周期和CD分子等方法研究MSCs的特性。结果证明MSCs在体外易扩增,传1—8代以内的细胞增殖能力无明显变化。未经诱导的MSCs大部分处于G_0/G_1期,G_0/G_1期的细胞所占百分比平均高于80%;骨髓基质细胞CD29(+),CD11b(-),CD90(+)。本实验应用复合诱导因子BME,RA,FSK,bFGF,PDGF,HRG体外连续诱导MSCs向Schwann细胞样细胞分化。诱导分化后采用免疫荧光细胞化学染色,流式细胞仪,Western Blot和逆转录PCR方法分别检测检测Schwann细胞的标记蛋白S100蛋白及其mRNA的表达。结果发现诱导分化后的MSCs形态上发生了类似Schwann细胞样细胞的变化。MSCs在诱导过程中S100的mRNA有上升的趋势;通过免疫荧光方法观察到诱导后MSCs表达S100蛋白增强;我们又通过Western Blot和流式细胞仪进一步定量检测到MSCs表达S100蛋白增高。又因为大多数的研究观察了细胞形态以及几种神经相关蛋白的免疫反应,也有报道用基因组学方法分析MSCs诱导过程中基因谱的表达变化。目前尚未见报道用蛋白质组学方法分析MSCs在诱导过程中蛋白谱的表达变化。我们进一步利用蛋白质组学技术分析MSCs向Schwann细胞样细胞诱导分化过程中蛋白谱的表达变化。我们采用2-DE技术分离未诱导和诱导分化过程中MSCs总蛋白,应用基质辅助激光解吸电离飞行时间质谱得到相应的肽质量指纹图谱,搜索数据库分析差异蛋白质点。我们所得到的MSCs蛋白谱有792±23个蛋白点,通过PDQuest软件分析未诱导MSCs蛋白谱和诱导分化过程中MSCs蛋白谱,初步分析发现有74个蛋白质的表达发生了明显的变化(p＜0.05),其中43个蛋白表达上调,31个蛋白表达下调。我们通过质谱分析并进行网上数据库搜索匹配,初步分析发现这些蛋白主要包括骨架和结构蛋白(tubulin alpha,vimentin,brain-specific alpha actinin 1 isoform等)、生长因子类的蛋白(ciliary neurotropic factor,brain-derived neurotrophic factor等)、代谢相关蛋白及酶类(UDP-glucose dehydrogenase,eph and elk-related kinase等)、伴侣蛋白(heat shockprotein等)、受体类蛋白(Laminin receptor 1,Ly49 stimulatory receptor 3等)、细胞周期蛋白(p27,RAS p21 protein activator 1等)、钙结合蛋白(nucleobindin 1,non musclecaldesmon等)以及其它蛋白等。总之,我们的实验结果表明MSCs在诱导分化后形态上发生了类似Schwann细胞样细胞的变化。未经诱导分化的MSCs表达少量S100蛋白,MSCs在这种体外条件诱导分化的过程中表达S100蛋白有明显的增加,而且在诱导分化过程中S100mRNA的表达有上升的趋势。 MSCs体外条件诱导分化过程中有较多蛋白表达发生变化;其中与神经细胞和神经胶质细胞相关蛋白:BDNF,CNTF,ILGF,pleiotrophin,FGF,GFAP,synaptophysin等在MSCs体外条件诱导分化过程中表达明显上调;本研究从蛋白质水平为MSCs体外向Schwann细胞样细胞条件诱导提供了新的研究资料。
[Abstract]:So far, the repair of peripheral nerve defects remains one of the most difficult problems to be solved in the field of clinical surgery. If the distance between the two ends is too far, it affects the growth of the regenerative axon. It is difficult to promote the development of neural tissue engineering research since the.20 century, especially the structure of "tissue engineering nerve" composed of scaffold materials and extracellular matrix, seed cells, and inducing and promoting growth factors. It provides new hope for people to search for nerve substitutes to repair peripheral nerve defects.
Schwann cells are the most important and most often used seed cells in the neural tissue engineering. But because the source of autologous Schwann cells is limited and it is not easy to proliferate, the allogenic Schwann cells will cause immune rejection. Therefore, the application of the "tissue engineering nerve" to the clinical study of the Schwann cell is still difficult to overcome. Bone marrow stromal cells MSCs (bone marrow stromal cells) has a wide range of sources, which can be expanded rapidly in a certain culture condition, with self renewal, active proliferation and multidirectional differentiation potential, and autologous transplantation overcomes the problems of ethical and immune rejection. As an ideal seed cell, it stands out.
In recent years, some in vitro studies have shown that MSCs can express the markers of neurons under the appropriate induction conditions. These studies have shown that MSCs has morphologically similar morphologic changes in neurons after induction of differentiation and can express specific proteins in neural cells, so these researchers think that MSCs sends to nerve cells. But there are different points of view, but there are different points of view. Some researchers believe that the differentiation of MSCs in the body is the process of fusion with the host's nerve cells. Some studies think that MSCs does not really differentiate, and the change of cell morphology is only due to the effect of inducer chemical stimulation to induce cell contraction and cell bone. It is a hot topic in the field of stem cell research that MSCs has potential to differentiate into neural lineage.
Our previous studies have shown that after MSCs transplantation in rats to the injured part of the sciatic nerve, part of the body can differentiate into Schwann cell like cells and express the S100 protein. The transplantation of MSC to the body is beneficial to the regeneration of the nerve. In order to further analyze the Schwann cell marker protein S10 in the process of MSC to Schwann fine cell differentiation. The change of expression of 0 protein is true. In our experiment, the morphologic changes of MSCs and the expression of S100B protein in Schwann cells during the induction of rat bone marrow stromal cells were investigated.
We isolated and cultured from the femur and tibia of adult SD rats and cultured MSCs. to detect cell viability by MTT method. FCM detected cell cycle and CD molecular methods to study the properties of MSCs. The results showed that MSCs was easily amplified in vitro, and the cell proliferation ability within 1 to 8 generations was not significantly changed. The uninduced MSCs was mostly in G_0/G_1 phase, G_0/G_1. The percentage of cells in the period was higher than 80%, bone marrow stromal cells CD29 (+), CD11b (-), CD90 (+).
This experiment used compound inducer BME, RA, FSK, bFGF, PDGF, HRG to induce MSCs to differentiate into Schwann cell like cells in vitro. Immunofluorescent cytochemical staining, flow cytometry, Western Blot and reverse transcription PCR were used to detect the expression of marker protein and expression of Schwann fine cell after induction of differentiation. The morphology of MSCs after induction of differentiation was similar to that of Schwann cell like cells. The mRNA of S100 increased in the induction process, and the MSCs expression of S100 protein was enhanced by immunofluorescence. We also quantified the MSCs expression S100 protein by Western Blot and flow cytometry.
Because most studies have observed the cell morphology and the immune responses of several nerve related proteins, it is also reported that the genomics method is used to analyze the changes in the expression of gene spectrum during the induction of MSCs. There is no report of proteomic analysis of the changes in the expression of egg white spectrum during the induction of MSCs. Analysis of the protein expression changes during the induction of differentiation of MSCs to Schwann cell like cells by white matter technology. We used 2-DE technology to separate MSCs total protein in the process of uninduced and induced differentiation. The corresponding peptide mass fingerprints were obtained by using matrix assisted laser desorption ionization time of flight mass spectrometry, and the differential protein was searched by database to analyze the differential protein. The MSCs protein spectrum has 792 + 23 protein points, and the PDQuest software is used to analyze the uninduced MSCs protein spectrum and the MSCs protein spectrum during the induction of differentiation. The preliminary analysis shows that the expression of 74 proteins has changed significantly (P < 0.05), of which 43 protein tables are up and 31 proteins are down regulated. Analysis and matching of online database search, preliminary analysis found that these proteins mainly include skeleton and structural proteins (tubulin alpha, vimentin, brain-specific alpha actinin 1 isoform, etc.), the protein of growth factor class (ciliary neurotropic factor, brain-derived neurotrophic), metabolic related proteins and enzymes Ose dehydrogenase, Eph and Elk-related kinase etc., chaperone protein (heat shockprotein, etc.), receptor class proteins (Laminin receptor 1, Ly49 stimulatory), cyclin protein (1, etc.), calcium binding protein (1, etc.), and other proteins.
In conclusion, our experimental results showed that the morphology of MSCs was similar to that of Schwann cell like cells after the induction of differentiation. The expression of S100 protein was obviously increased during the induction of differentiation without the MSCs expression of a small amount of S100 protein in the undifferentiated MSCs, and the expression of S100mRNA in the induced differentiation process was higher. The trend of rising.
The expression of more protein in the process of MSCs induced differentiation in vitro was changed, and the expression of BDNF, CNTF, ILGF, pleiotrophin, FGF, GFAP, synaptophysin in the process of differentiation in vitro was obviously up-regulated in the process of induction and differentiation of MSCs in vitro; this study was from protein level to Schwann cell like in vitro. Cell condition induction provides new research data.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329

【共引文献】